Prostaglandin D2 (PGD2) is a naturally occurring prostaglandin that has been shown to be a mediator in allergic and inflammatory disorders (Spik, I., Brenuchon, C., Angeli, V., et al. J. Immunol., 2005, 174, 3703-3708; Urade, Y., Hayaishi, O. Vitamin and Hormones, 2000, 58, 89-120). PGD2 is formed from arachidonic acid by reactions catalyzed by prostaglandin endoperoxide synthase (cyclooxygenase, COX) and PGD synthase (PGDS). COX catalyzes two consecutive reactions, dioxygenation of arachidonic acid to PGG2 and peroxidation of PGG2 to PGH2, the common precursor of prostanoids (Aritake, K., Kado, Y., Inoue, T., Miyano, M., Urade, Y. J. Biol. Chem., 2006, 281, 15277-15286). PGH2 metabolism leads to PGE9. PGD2, PGF2, PGI2 and thromboxane A2 (TXA2).
Two distinct types of prostaglandin D synthases are involved in PGD2 production: lipocalin-type PGDS (L-PGDS) and hematopoietic PGDS (H-PGDS). L-PGDS and H-PGDS differ with respect to primary amino acid sequence, cellular localization and tertiary structure. L-PGDS, also known as β-trace, is localized in the central nervous system, male genital organs, and heart and is involved in the regulation of sleep and pain (Aritake et al., 2006). H-PGDS is associated with allergic and inflammatory reactions due to its localization in mast cells, Th2 cells, microglia, necrotic muscle fibers and apoptotic smooth muscle cells (Aritake et al., 2006). H-PGDS requires glutathione for activity and belongs to the sigma-class of glutathione S-transferases (Kanaoka, Y., Fujimora, K., Kikuno, R., et al., Eur. J. Biochem., 2000, 267, 3315-3322; Kanaoka, Y., Ago, H., Inagaki, E., et al., Cell, 1997, 90, 1085-1095; Urade, Y., Fujimoto, N., Ujihara, M., et al., J. Biol. Chem., 1987, 262(8), 3820-3825). Two well-known H-PGDS inhibitors, namely HQL-79 and Tranilast, have both been shown to reduce PGD2 levels in guinea pig lung tissues chronically treated with the inhibitors (Matsushita, N., Hizue, M., Aritake, K., Hayashi, K., Takada, A., Mitsui, K., Hayashi, M., Hirotsu, I., Kimura, Y., Tani, T., Nakajima, H. Jpn. J. Pharmacol., 1998, 78, 1-10). Both inhibitors possess micromolar IC50 values against the synthase in known in vitro assays. Recent patent application publications describe pyrimidine amide compounds (U.S. Appn. No. 2008/0207651 to Blake et al., entitled “Heterocyclic Compounds Useful in Treating Disease and Conditions; U.S. Appn. No. 2008/0227782 to Aldous et al., entitled “Pyrimidine Amide Compounds as PGDS Inhibitors”) and pyridine amide compounds (U.S. Appn. No. 2008/0146569 to Blake et al., entitled “Nicotinamide Derivatives”) as H-PGDS inhibitors with nanomolar IC50s.
Currently known in vitro H-PGDS inhibition assays typically quantify PGD2 production using PGD2 enzyme immunoassays (EIAs), fluorescence polarization enzyme immunoassays (FPIAs), or the corresponding radioimmunoassay (RIAs) in order to determine a compound's or agent's ability to modulate PGD2 production. These functional assays utilize the unstable prostanoid precursor PGH2 as the H-PGDS substrate. PGH2 can non-enzymatically convert to PGD2 and PGE2 and thus assays that measure PGD2 production from PGH2 must employ cumbersome and precisely-timed reaction and quenching sequences in order to minimize non-enzymatic production of PGD2. These assays are not amenable to high-throughput screening (HTS).
Other in vitro H-PGDS assays involve the use of glutathione S-transferase (GST) substrates such as chloro-dinitrobenzene (CDNB) or monochlorobimane (MCB), in which the conjugation of glutathione (GSH) to CDNB or MCB is measured by colorimetry or fluorometry, respectively. (Greig, G. M., Masse, F., Nantel, F., et al., J. Allergy Clin. Immunol., 2006, 117 (Suppl. 2), S66). A limitation of this assay could be that it would select for inhibitors that can also inhibit endogenous GSTs. GSTs are important detoxifying enzymes and are known to play significant role in xenobiotic metabolism and inhibiting these enzymes could have toxicological implications downstream. Another potential limitation inherent in GST assays is the general bias of these assays toward compounds that may conjugate directly with GSH but do not bind to H-PGDS in eukaryotic cells. Finally, the ability of CDNB and MCB to conjugate with GSH non-enzymatically, can cause low signal-to-noise ratios and narrow dynamic range in these assays.
A known cell-based assay that simultaneously measures potency, specificity, and cytotoxicity of H-PGDS modulators involves stimulation of the arachidonic acid cascade in any mammalian cell line in which human PGD2 is expressed as described in WO 2006/015195 to Yang et al., entitled “Method for Determining the Potency, Specificity, and Toxicity of Hematopoietic D2 Synthase.”
Fluorescence polarization (FP) assays provide advantages in the study of protein-ligand binding over conventional methods such as those described above. FP assays allow real-time measurements, avoid the use of radioactive materials, are homogeneous, typically comprise fewer steps (require no washing step), and may possess sub-nanomolar detection limits. FP assays are currently used in drug discovery and are routinely converted to high-throughput screening (HTS) format (Burke, T. J., Loniello, K. R., Beebe, J. A., Ervin, K. M. Comb. Chem. High Throughput Screen., 2003, 6(3), 183-194).
Fluorescence is one of a number of phenomena generally referred to as luminescence. Fluorescence is a luminescence in which the molecular absorption of a photon of a specific wavelength (excitation wavelength) triggers the emission of a photon of longer (lower-energy) wavelength, while the remainder of the absorbed energy is usually translated into increased molecular motion or thermal energy. The molecular component of a fluorescent substance that causes it to fluoresce is called the fluorophore. The photon of a particular frequency (νex) promotes a fluorophore from its ground-state (S0) into an excited state (S1):S0+hνex→S1 (h=Planck's constant)
Fluorescence occurs with the transition of a fluorophore excited-state electron to its ground state, which is accompanied by the emission of a longer-wavelength, lower-frequency photon (νem):S1→hνem+S0 
Fluorescence polarization operates on the principle that when a fluorescent molecule is excited with polarized light, light is emitted in the same polarized plane if the excitation lifetime is less than the time it takes for the molecule to tumble out of this plane. Should the high-energy state exist longer than the time it takes for the molecule to tumble out of the excitation plane, light is emitted in a plane different from the excitation plane, which results in the detection of a relatively depolarized signal. Very large, high-mass molecules are less likely to rotate out of the excitation plane prior to emission and are therefore more likely to emit highly polarized light and produce a strong polarization signal. Smaller molecules are more likely to tumble out of the excitation plane prior to relaxation and emission and therefore provide relatively depolarized (relative to the excitation plane) emitted light and a weaker FP signal. To evaluate the polarization two measurements are needed: the first using a polarized emission filter parallel to the excitation filter (S-plane) and the second with a polarized emission filter perpendicular to the excitation filter (P-plane). The fluorescence polarization response is given as mP (milli-Polarization) level and is obtained from the equation:Polarization (mP)=1000×[S−(G×P)]/[(S+(G×P)]where S and P are background subtracted fluorescence count rates and G (grating) is an instrument and assay dependent factor. The rotational speed of a molecule is dependent on the size of the molecule, temperature and viscosity of the solution. Fluorescein, rhodamine, and DyLight™ 633 have fluorescence lifetimes suitable for the rotation speeds of molecules in bio-affinity assays such as receptor-ligand binding assays. The basic principle is that the detection analyte is small and rotates rapidly (low polarization). When the detection analyte binds to the larger molecule (enzyme), its rotation slows down considerably (polarization changes from low to high polarization).